Before investigators can effectively use immunotoxins (chimeric molecules composed of an antibody molecule linked to a toxin molecule) in in vivo or in vitro protocols it is essential that these researchers have an understanding of the essential molecular features necessary for optimum conjugate cytotoxicity. To that end the long-range objectives of this research are: (1)\to determine the requisite features of the antibody, the toxin and the coupling reagent necessary for optimum cytotoxicity, (2)\to characterize the effects of antigen density and its modulation of immunotoxin internalization, and (3)\to understand the mechanisms of immunotoxin translocation and processing. More specifically, our immediate goals are: (1)\to construct ricin (native and modified) and ricin A chain-containing immunotoxins with a pan-T-cell antibody using cleavable and non-cleavable cross-linking reagents and to compare their efficacy in vitro, and (2)\to characterize the involvement of actin, calmodulin and transglutaminase in conjugate internalization to determine if immunotoxins are internalized via receptor-mediated endocytosis, and to clarify the putative role of proton gradients in conjugate internalization and processing. While some aspects of conjugate synthesis (e.g., optimum toxin-to-antibody ratio) and the cytotoxicity assays have not yet been fully determined, most of the methodologies (protein fluorescenation, iodination, and the isolation of clathrin coated vesicles, for example) are well characterized and technically feasible. The accomplishment and critical evaluation of the specific aims described above should allow oncologists and other investigators to synthesize and employ more effective immunotoxins in chemotherapeutic protocols with both in vitro and in vivo applications.